The detection, analysis, transcription, and amplification of nucleic acids are some of the most important procedures in modern molecular biology. The application of such procedures for nucleic acid analysis is especially important in the investigation of gene expression, diagnosis of infectious agents or genetic diseases, the generation of cDNA, and analysis of retroviruses, to name but a few applications. The reverse transcription of RNA, followed by polymerase chain reaction (PCR) cDNA amplification, commonly referred to as RT-PCR or RNA-PCR, has become widely used for the detection and quantification of nucleic acid targets and is particularly important for viral gene analysis.
The study of oncoviruses and their roles in the pathogenesis of carcinomas can have important implications in the diagnosis and treatment of cancer. However, variability in the detection of oncoviruses can make these studies challenging. Sensitivity and specificity of the detection method are key concerns. Earlier detection can be achieved using molecular biology techniques to detect oncoviral nucleic acids in samples that have not undergone seroconversion, and these techniques are applicable to viruses that cannot be propagated in tissue culture. Traditionally, immunological detection methods have been used to detect the presence of oncoviruses, but these methods have several drawbacks. In the case of Human Papillomavirus (HPV), which can cause cervical cancer, detection is difficult due to low expression of early viral proteins and a lack of sensitive and specific high-quality antibodies that can discriminate HPV types (Villa and Denny, International Journal of Gynecology and Obstetrics, 94(Suppl. 1):S71-S80 (2006)). Results can also be misleading or inconclusive when a sample exhibits residual antibody levels due to an infection that may have happened months or years prior to sample collection, as in the case of the Epstein-Barr Virus (EBV), which is associated with Hodgkin's lymphoma and other carcinomas (National Center for Infectious Diseases. “Epstein-Barr Virus and Infectious Mononucleosis,” CDC. www.cdc.gov/ncidod/diseases/ebv (November 2010)). PCR-based detection of viral nucleic acids not only has the advantage of being highly specific due to sequence specific primer binding, it is also less cumbersome and more sensitive than other hybridization techniques, such as the Northern and Southern blots, due to its ability to amplify shorter nucleic acid fragments. Sequence-specific probe binding adds an additional layer of specificity to real-time PCR and has the additional advantage of providing viral load information.
RT-PCR typically involves two separate molecular syntheses: (i) the synthesis of cDNA from an RNA template; and (ii) the replication of newly synthesized cDNA through PCR amplification. RT-PCR can be performed by one-step (or coupled) RT-PCR methods using two or more enzymes, in which at least two separate enzymes (e.g., a reverse transcriptase and a polymerase) are employed for initial cDNA synthesis and subsequent amplification, respectively.
In one-step RT-PCR, reverse transcription and PCR amplification are combined into a single reaction mixture which provides numerous advantages over two-step RT-PCR (where the synthesis and amplification steps are performed using two different or separate reactions). One-step RT-PCR requires less handling of the reaction mixture reagents and nucleic acid products than two-step RT-PCR (e.g., opening of the reaction tube for component or enzyme addition in between the two reaction steps), and is therefore less labor intensive and time consuming. One-step RT-PCR also allows for less sample to be used if necessary, and reduces the risk of contamination (Sellner and Turbett, Biotechniques 25:234-238 (1998)).
The use of one-step RT-PCR methods have some drawbacks, however. For example, individual optimization of the ratio of reverse transcriptase to DNA polymerase is usually not practicable for ready-to-use compositions or kits for one-step RT-PCR. Several reports have also documented interference between reverse transcriptase and DNA polymerase when used in combination in a single tube RT-PCR reaction resulting in low sensitivity or lack of results (Sellner, L. N., et al., Nucl. Acids Res. 20:1487-1490 (1992)).
Moreover, samples from which viral nucleic acids are extracted often contain additional compounds that are inhibitory to PCR. Humic acid in soil and feces, hematin in blood, immunoglobin G in serum, and various blood anticoagulants, like heparin and citrate, are all examples of such inhibitors. Such inhibitors may not be completely removed during the nucleic acid extraction and purification process, thus negatively impacting downstream PCR amplification, as reflected by an increase in Ct (i.e., threshold cycle) and decrease in dRn (i.e., difference in normalized reporter signal) when assayed by real time PCR.
A high Ct coupled with low dRn usually indicates low target nucleic acid concentration in reactions for quantitative PCR (qPCR) and reverse transcriptase-qPCR (RT-qPCR) applications. A reaction that exhibits reduced or no amplification indicates that the target nucleic acid is absent, or present in such small amounts that it is not detectable. A reaction that contains detectable amounts of target, but is inhibited by the presence of PCR inhibitors may show an artificially high Ct and low dRn, which can lead the user to believe that the amount of target nucleic acid is less than the actual amount present. If the level of inhibition is severe enough, the reaction may fail to amplify completely, thus leading to a false-negative result.
Because of the importance of nucleic acid synthesis applications to the fields of molecular biology and cellular biology, a one-step RT-PCR system, in the form of a generalized ready-to-use composition, which exhibits high sensitivity, is not restricted by the amount of sample, reduces the amount of practitioner manipulation, minimizes the risks of contamination, minimizes the expense of reagents, and maximizes the amount of nucleic acid end product, is needed in the art. In addition, a method to reduce or eliminate the negative effects of PCR inhibitors, especially when analyzing viral targets where sample sources often contain such inhibitors, is necessary to ensure accurate results.